1. Field of the Invention
This invention relates to a monoclonal antibody capable of specifically binding to human VEGF receptor Flt-1 which is useful the diagnosis or treatment of diseases in which their morbid states progress by abnormal angiogenesis, such as proliferation or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity and psoriasis; a hybridoma capable of producing the antibody; a method for immunologically detecting human VEGF receptor Flt-1 using the monoclonal antibody; and a diagnostic method and a therapeutic method for diseases, such as solid tumor, rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, psoriasis and the like, using the monoclonal antibody.
2. Brief Description of the Background Art
Angiogensis plays an important role in the individual development and construction of tissues in vertebrates, is directly involved in the formation of the corpus luteum during the sexual cycle, transient proliferation of the uterine endometrium and formation of the placenta in mature individuals (females). With regard to pathological states, angiogenesis is involved in the proliferation or metastasis of solid tumors and formation or acceleration of morbidity in diabetic retinopathy and rheumatoid arthritis (J. Biol. Chem., 267, 10931 (1992). Angiogenesis occurs by the secretion of an angiogenesis factor and involves process of a tube formation and producing a new blood vessel. During this process, the basement membrane and interstitum are destroyed by a protease secreted from endothelial cells of an existing blood vessel around the secreted angiogenesis factor, followed by subsequent wandering and proliferation of vascular endothelial cells (J. Biol. Chem., 267, 10931, (1992)). Factors which induce angiogenesis include vascular permeability factor (hereinafter xe2x80x9cVPFxe2x80x9d) and vascular endothelial growth factor (hereinafter xe2x80x9cVEGFxe2x80x9d) (hereinafter xe2x80x9cVPF/VEGFxe2x80x9d). These factors are considered the most important factors in pathological and non-pathological angiogenesis (Advances in Cancer Research, 67, 281 (1995)). VPF/VEGF is a protein having a molecular weight of about 40,000 constituted by homodimers, which had been reported to be independent molecules as vascular permeability factor (VPF) in 1983 (Science, 219, 983 (1993)) and as vascular endothelial growth factor (VEGF) in 1989 (Biochem. Biophys. Res. Comm., 161, 851 (1989)), but it has been revealed as the results of cDNA cloning that they are the same substance (Science, 246, 1306 (1989); Science 246, 1309 (1989)) (hereinafter, the term xe2x80x9cVPF/VEGFxe2x80x9d is recited as xe2x80x9cVEGFxe2x80x9d). Beyond the activity of VEGF upon vascular endothelial cells described above, VEGF has also been shown to have a growth enhancing activity (Biochem. Biophys. Res. Comm., 161, 851 (1989)), a migration enhancing activity (J. Immunology, 152, 4149 (1994)), a metalloprotease secretion enhancing activity (J. Cell Physiol., 153, 557 (1992)), a urokinase and tPA secretion enhancing activity (Biochem, Biophys. Res. Comm., 181, 902 (1991)), and the like. Furthermore, VEGF has been shown to have an angiogenesis enhancing activity (Circulation, 92 suppl II, 365 (1995)), a vascular permeability enhancing activity (Science, 219, 983 (1983)), and the like as its in vivo activities. It has been reported that VEGF is a growth factor having extremely high specificity for vascular endothelial cells (Biochem. Biophys. Res. Comm., 161, 851 (1989)) and that four proteins having different molecular weight are present due to alternative splicing of mRNA (J. Biol. Chem., 267, 26031 (1991)).
Among diseases accomplished by angiogenesis, it has been reported that VEGF plays an important role in the proliferation or metastasis of solid tumors and formation of morbid states of diabetic retinopathy and rheumatiod arthritis. With regard to solid tumors, production of VEGF in a number of human tumor tissues has been reported, such as in renal carcinoma (Cancer Research, 54, 4233 (1994)), breast cancer (Human Pathology, 26, 86 (1995)), brain tumor (J. Clinical Investigation, 91, 153 (1993)), gastrointestinal cancer (Cancer Research, 53, 4727 (1993)), ovarian cancer (Cancer Research, 54, 276 (1994)), and the like. Also, results of a study on the correlation between VEGF expression quantity in tumors and survival ratio of patients in patients with breast cancer have revealed that tumor angiogenesis is more active in tumors expressing high levels of VEGF than low VEGF expression tumors and that the survival ratio is lower in breast cancer patients having high VEGF expression tumors than breast cancer patients having low VEGF expression tumors (Japanese J. Cancer Research, 85, 1045 (1994)). It has been reported also that an anti-VEGF monoclonal antibody inhibited tumor growth in a xenograft model test system in which a human tumor was transferred into nude mice by subcutaneous transplantation (Nature, 362, 841 (1993)). Also, it has been reported that, in a metastatic cancer model of a human tumor in nude mice, an anti-VEGF monoclonal antibody inhibited metastasis of the tumor (Cancer Research, 56, 921 (1996)). Additionally, since a high concentration of VEGF was detected in human carcinomatous pleural perfusions and ascites, the possibility that VEGF is a major factor involved in the retention of pleural perfusions and ascites has been suggested (Biochimica et Biophysica Acta, 1221, 211 (1944)).
In diabetic retinopathy, abnormal angiogenesis causes retinal detachment and hemorrhage of the vitreous body, resulting in blindness, and it has been reported that angiogenesis in diabetic retinopathy and the expression level of VEGF in the patient""s eye balls are positively correlative (New England J. Medicine, 331, 1480 (1994)). Also, it has been reported that angiogenesis in a monkey retinopathy model is inhibited when the VEGF activity is inhibited by the intraocular administration of an anti-VEGF neutralizing monoclonal antibody (Arch. Ophthalmol., 114, 66 (1996)).
Progress in the morbid states of rheumatoid arthritis (destruction of bone and cartilage) is accompanied by angiogenesis, and it has been reported that a high concentration of VEGF is contained in the synovial fluid of patients with rheumatoid arthritis and that macrophages in joints of patients with rheumatoid produce VEGF rheumatoid arthritis (Journal of Immunology, 152, 4149 (1994); J. Experimental Medicine, 180, 341 (1994)).
VEGF receptors have been reported. These include fms-like tyrosine kinase (referred to as xe2x80x9cFlt-1xe2x80x9d hereinafter) (Oncogene, 5, 519 (1990); Science, 255, 989 (1992)) and kinase insert domain-containing receptor (referred to as xe2x80x9cKDRxe2x80x9d hereinafter) (WO 92/14748; Proc. Natl. Acad. Sci., USA, 88, 9026 (1992); Biochem. Biophys. Res. Comm. 187, 1579 (1992); WO 94/11499), which belong to the receptor type tyrosine kinase family. Each of Flt-1 and KDR is a membrane protein of 180 to 200 kilodalton in molecular weight which has an extracellular domain consisting of 7 immunoglobulin-like regions and an intracellular domain consisting of a tyrosine kinase region. It has been reported that VEGF specifically binds to Flt-1 and KDR at Kd values of 20 pM and 75 pM and that Flt-1 and KDR are expressed in vascular endothelial cells in a specific manner (Proc. Natl. Acad. Sci., USA, 90, 7533 (1993); Proc. Natl. Acad. Sci., USA, 90, 8915 (1993)). With regard to Flt-1 in various diseases, it has been reported that, in comparison with vascular endothelial cells in normal tissues, expression of Flt-1 mRNA increases in tumor vascular endothelial cells of human glioblastoma tissues (Nature, 349, 845 (1992)) and tumor vascular endothelial cells of human digestive organ cancer tissues (Cancer Research, 53, 4727 (1993)). Additionally, it has been reported that expression of Flt-1 mRNA is observed by in situ hybridization in vascular endothelial cells of joints of patients with rheumatoid arthritis (J. Experimental Medicine, 180, 341 (1994)). These results strongly suggest that a VEGF/VEGF receptor Flt-1 system plays an important role in tumor angiogenesis. Although it has been reported that VEGF binds to Flt-1 and the intracellular domain is auto-phosphorylated (Science, 255, 989 (1992)), the detailed function of the receptor mechanism is still unclear. However, it has been discovered that knock out mice which the Flt-1 gene was destroyed die after a fetal age of 8.5 to 9.5 days due to abnormal blood vessel construction caused by abnormal morphology of vascular endothelial cells during blood island formation in the early stage of development and subsequent angiogenesis. This had led to an assumption that Flt-1 has a function essential for the tube formation of vascular endothelial cells in angiogenesis (Nature, 376, 66 (1995)).
In view of the above, it is expected that an antibody which can inhibit biological activities of VEGF through its binding to VEGF receptor Flt-1 will be useful for the diagnosis or treatment of diseases in which their morbid states progress by abnormal angiogenesis, such as proliferation or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity and psoriasis. However, an anti-VEGF receptor Flt-1 monoclonal antibody which can detect cells in which VEGF receptor Flt-1 is expressed and anti-VEGF receptor Flt-1 monoclonal antibody which can inhibit biological activities of VEGF has not been described in the art.
Concern has been described toward the development of a method which is useful for the diagnosis or treatment of diseases in which their morbid states progress by abnormal angiogenesis, such as proliferation or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity and psoriasis. Although nothing has been reported on the anti-human VEGF receptor Flt-1 monoclonal antibody, it is considered that detection of the regions of angiogenesis and inhibition of angiogenesis by the use of an anti-human VEGF receptor Flt-1 monoclonal antibody will be useful for the diagnosis and treatment of these diseases.
The present invention relates to a monoclonal antibody which specifically reacts with human VEGF receptor Flt-1; a monoclonal antibody which recognizes an epitope present in a region of amino acids 1 to 750, especially 1 to 338, of the N-terminal sequence of human VEGF receptor Flt-1 signal and receptor protein; (SEQ ID NO: 5 and 6) a monoclonal antibody which specifically reacts with human VEGF receptor Flt-1 by immunocyte staining; and a monoclonal antibody which inhibits binding of human VEGF to human VEGF receptor Flt-1 and inhibits biological activities of human VEGF.
Furthermore, the present invention relates to monoclonal antibody KM1730 belonging to mouse IgG1 subclass produced by hybridoma KM1730 (FERM BP-5697); monoclonal antibody KM1731 belonging to mouse IgG2a subclass produced by hybridoma KM1731 (FERM BP-5718); monoclonal antibody KM1732 belonging to mouse IgG1 subclass produced by hybridoma KM1732 (FERM BP-5698); monoclonal antibody KM1748 belonging to mouse IgG2b subclass produced by hybridoma KM1748 (FERM BP-5699); and monoclonal antibody KM1750 belonging to mouse IgG2b subclass produced by hybridoma KM1750 (FERM BP-5700).
Moreover, the present invention relates to hybridoma KM1730 (FERM BP-5697) which produces monoclonal antibody KM1730; hybridoma KM1731 (FERM BP-5718) which produces monoclonal antibody KM1731; hybridoma KM1732 (FERM BP-5698) which produces monoclonal antibody KM1732; hybridoma KM1748 (FERM BP-5699) which produces monoclonal antibody of KM1748; and hybridoma KM1750 (FERM BP-5700) which produces monoclonal antibody KM1750.
Also, the present invention relates to a method for detecting a disease in which the morbid states progress by abnormal angiogenesis, comprising the steps of: (a) separating human cell or a crushing solution thereof, tissue or a crushing solution thereof, serum, pleural fluid, ascites fluid, or ocular fluid to prepare a sample, (b) reacting the separated sample prepared in the step (a) with the monoclonal antibody of the present invention, (c) further reacting the reacted sample prepared in the step (b) with a labeled anti-mouse IgG antibody or binding fragment, and (d) measuring or observing the labeled sample prepared in the step (c); a method for immunologically detecting human VEGF receptor Flt-1, comprising the step of reacting the human VEGF receptor Flt-1 with the monoclonal antibody of the present invention; a method for immunologically detecting cells in which human VEGF receptor Flt-1 is expressed on the surface thereof, comprising the step of reacting the human VEGF receptor Flt-1 with the monoclonal antibody of the present invention; and a method for detecting and determining soluble human VEGF receptor Flt-1, comprising the step of reacting the human VEGF receptor Flt-1 with the monoclonal antibody of the present invention.
Still furthermore, the present invention relates to a method for preventing or treating a disease, comprising the step of administering to human or animal in need of such prevention or treatment an effective amount of the monoclonal antibody of the present invention; a method for inhibiting binding the human VEGF to human VEGF receptor Flt-1, comprising the step of reacting the human VEGF receptor Flt-1 with the monoclonal antibody of the present invention; and a method for inhibiting biological activities of human VEGF, comprising the step of reacting human VEGF receptor Flt-1 with the monoclonal antibody of the present invention.
Still moreover, the present invention relates to a composition comprising the monoclonal antibody of the present invention and a diagnostic or pharmaceutical acceptable carrier.